ES2580108T3 - Isoquinoline compounds - Google Patents

Abstract

Compound according to formula (I): ** Formula ** in which A is a substituted or unsubstituted isoquinolin-6-yl, in which the isoquinoline radical can be mono or disubstituted with halogen, cyano, nitro or C1-alkyl -C4; R1, R2, R3, R4 and R5 are independently hydrogen, halogen, C1-C8-alkyl, alkoxy, phenoxy, -OR7, amino, nitro, cyano, aryl, C1-C4-alkylaryl, heteroaryl, C1-C4-heteroaryl alkyl , carbonylamino, thioalkyl, sulfonyl, sulfonylamino, acyl or carboxyl; R7 is C1-C4-alkyl, aryl, heteroaryl, C1-C4-alkyl-aryl or C1-C4-alkyl-heteroaryl; X is; and R6 is CH2 or CH (C1-C4 alkyl).

Description

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DESCRIPTION

Isoquinoline compounds

The present invention relates to isoquinoline compounds that can affect the action of receptor kinases coupled to G proteins in a cell and that are useful as therapeutic agents or with therapeutic agents. In particular, these compounds are useful in the treatment of eye diseases or eye disorders such as glaucoma.

A variety of hormones, neurotransmitters and other biologically active substances control, regulate or adjust the functions of living bodies through specific receptors located in cell membranes. Many of these receptors mediate intracellular signal transmission by activating guanine nucleotide binding proteins (G proteins) to which the receptor is coupled. Such receptors are generically referred to as G-protein coupled receptors (GPCRs) and include, among others, a-adrenergic receptors, p-adrenergic receptors, opioid receptors, cannabinoid receptors and prostaglandin receptors.

The receptors coupled to G proteins play an important role in the regulation of various physiological functions. For example, GPCRs have been implicated in several pathological conditions including: cardiac indications such as angina pectoris, essential hypertension, myocardial infarction, supraventricular and ventricular arrhythmias, congestive heart failure, atherosclerosis, renal failure, diabetes, respiratory indications such as asthma , chronic bronchitis, bronchospasm, emphysema, obstruction of the respiratory tract, indications of upper respiratory tract such as rhinitis, seasonal allergies, inflammatory disease, inflammation in response to injury, rheumatoid arthritis, inflammatory bowel disease, glaucoma, hypergastrinemia, indications gastrointestinal disorders such as acid / peptic disorder, erosive esophagitis, gastrointestinal hypersecretion, mastocytosis, gastrointestinal reflux, peptic ulcer, Zollinger-Ellison smdrome, pain, obesity, bulimia nervosa, depression, obsessive-compulsive disorder, organ malformations (for example, cardiac malformations), neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease, multiple sclerosis, Epstein-Barr infection and cancer.

The balance between the initiation and deactivation of the intracellular signal, called desensitization, regulates the intensity and duration of the response of the receptors to stimuli such as agonists. The desensitization of GPCRs occupied by agonists results from their phosphorylation by specific kinases called G-protein-coupled receptor kinases (GRK) and the subsequent binding of arresting proteins to phosphorylated receptors. Arrests are a family of intracellular proteins that bind activated GPCRs, including those that have been activated by agonists, and especially those that have been phosphorylated by receptor kinases coupled to G proteins. The binding of arrests prevents further stimulation of proteins. G and subsequent signaling routes. When desensitization occurs, the mediation or regulation of the physiological function mediated or regulated by the G-proteins to which the receptors are coupled is reduced or prevented. For example, when agonists are administered to treat a disease or condition by activating certain receptors, the receptors become desensitized from the action of the GRKs so that the administration of agonists may no longer result in therapeutic activation. of the appropriate receptors. At that point, the administration of the agonist no longer allows sufficient or effective control of or influence the disease or condition that is intended to be treated.

WO-A-2005020921 discloses compounds for modulating the enzymatic activity of protema kinases to modulate cellular activities such as proliferation, differentiation, programmed cell death, migration and chemoinvasion. Even more specifically, the invention provides compounds for modulating c-Kit kinase activity and methods of treating diseases mediated by e-Kit activity using the compounds and pharmaceutical compositions thereof.

EP-A-1550660 discloses compounds of the following formula (I) that have Rho kinase inhibitory activity.

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WO-A-2005035503 discloses an isoquinoline derivative that has Rho kinase inhibitory activity.

WO-A-0153274 discloses amide compounds that modulate and / or inhibit the activity of certain protein kinases. These compounds and pharmaceutical compositions containing them may mediate the transduction of tyrosine kinase signals to modulate and / or inhibit unwanted cell proliferation.

EP-A-0389995 discloses the use of isoquinoline derivatives of the following formula (I) for the

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treatment of glaucoma or ocular hypertension

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EP-A-0232569 discloses 1-benzyl-2- (N-substituted) -carbamoyl-tetrahydroisoquinolines that have an inhibitory effect on beta-adenylate cyclase.

G. Shankar et al. disclose that specific protein kinase inhibitors block the migration of Langerhans cells by inhibiting the release of interleukin-1 alpha; Immunology, vol. 96, No. 2, 1999, pages 230-235.

R. B. Penn et al. disclose the pharmacological inhibition of protema kinases in intact cells: the antagonism of the binding to beta-adrenergic receptors by H-89 reveals limitations in its usefulness; Journal of Pharmacology and Experimental Therapeutics, vol. 288, No. 2, 1999, pages 428-437.

In view of the role of GRKs in the desensitization of GPCRs, there is a need in the art for agents that prevent or reduce desensitization of GPCRs by controlling or inhibiting the action of the corresponding GRKs.

A compound according to formula (I) is provided:

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wherein A is a substituted or unsubstituted isoquinoline radical, in which the isoquinoline radical may be mono or disubstituted with halogen, cyano, nitro or C1-C4 alkyl;

R1, R2, R3, R4, and R5 are independently hydrogen; halogen; C1-C8 alkyl; alkoxy; phenoxy, -OR7; Not me; nitro; cyano; aryl; C1-C4-aryl alkyl; heteroaryl; C1-C4 alkyl-heteroaryl; carbonylamino; thioalkyl; sulfonyl; sulfonylamino; acyl; or carboxyl;

R7 is C1-C4 alkyl, aryl, heteroaryl, C1-C4 alkyl-aryl or C1-C4 alkyl-heteroaryl;

X is

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Y

R6 is CH2 or CH (C1-C4 alkyl).

In another embodiment of the invention, an isoquinoline compound according to formula (II) is provided:

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in which R1

R2 ’, R3’, R4 ’and R5’ are independently hydrogen; halogen; unsubstituted C1-C4 alkyl; Not me;

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nitro; cyano; carbonylamino; alkoxy; -O-R7; sulfonylamino; carboxyl; acyl; or thioalkyl; R 7 'is C 1 -C 4 alkyl, aryl, heteroaryl, C 1 -C 4 alkyl-aryl or C 1 -C 4 alkyl-heteroaryl;

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X ’is H i y

R6 'is CH2 or CH (C1-C4 alkyl).

In another embodiment of the invention, a compound according to formula (III) is provided:

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wherein R8 and R9 are independently hydrogen; halogen; unsubstituted C1-C4 alkyl; substituted C1-C4 alkyl; Not me; nitro; cyano; carbonylamino; alkoxy; phenoxy; benzyloxy; -O-R10; sulfonylamino; carboxyl; acyl or thioalkyl; Y

R10 is unsubstituted C1-C4 alkyl; substituted C1-C4 alkyl; substituted aryl; heteroaryl; substituted heteroaryl; C1-C4 alkaryl or C1-C4-heteroaryl alkyl;

wherein when R8, R9 or R10 is substituted, the substituent is selected from amino, cyano, halogen, alkoxy or hydroxyl; when R8, R9 or R10 is substituted aryl, the substituent is selected from amino, halogen, cyano, halogen or hydroxyl; when R8, R9 or R10 is substituted heteroaryl, the substituent is selected from halogen, cyano, nitro or C1-C4 alkyl.

In another embodiment of the invention, a pharmaceutical composition is provided, said pharmaceutical composition comprising:

a) an isoquinoline derivative according to the invention; Y

b) a carrier.

In another embodiment of the invention, an isoquinoline derivative is provided according to the invention for use in the treatment of a condition, in which the condition is selected from the group consisting of eye disease, bone disorder (such as osteoporosis), disease heart disease, liver disease, kidney disease, pancreatitis, cancer, myocardial infarction, gastric discomfort, hypertension, fertility control, nasal congestion, neurogenic bladder disorder, gastrointestinal disorder and dermatological disorder.

Preferred embodiments are set forth in the dependent claims.

Figure 1 is a graphical representation of the effect of a 60-minute pre-incubation of the GRK-2 inhibitor of Example 2 on the translocation of p-arrestin induced by ISO into p2wt using a Transfluor® assay.

Figure 2 is a graphical representation of the effect of a 60-minute pre-incubation of the GRK-2 inhibitor of Example 2 on the translocation of p-arrestin induced by ISO into p1wt using a Transfluor® assay.

Figure 3 is a graphical representation of the effect of a 60-minute pre-incubation of the GRK-2 inhibitor of Example 2 on the translocation of morphine-induced p-arrestine from the opioid receptor | i using a Transfluor® assay.

Figure 4 is a graphical representation of the effect of a 60-minute pre-incubation of the GRK-2 inhibitor of Example 3 on the translocation of ISO-induced p-arrestin into p2wt using a Transfluor® assay.

Figure 5 is a graphical representation of the effect of a 60-minute pre-incubation of the GRK-2 inhibitor of Example 3 on the translocation of p-arrestin induced by ISO into p1wt using a Transfluor® assay.

Figure 6 is a graphical representation of the effect of a 60-minute pre-incubation of the GRK-2 inhibitor of Example 3 on the translocation of morphine-induced p-arrestine from the opioid receptor using a Transfluor® assay.

Figure 7 is a graphical representation of the effect of a 60-minute pre-incubation of the GRK-2 inhibitor of

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Example 4 on translocation of ISO-induced p-arrestin into p2wt using a Transfluor® assay.

Figure 8 is a graphical representation of the effect of a 60-minute pre-incubation of the GRK-2 inhibitor of Example 4 on the translocation of ISO-induced p-arrestin in piwt using a Transfluor® assay.

Figure 9 is a graphical representation of the effect of a 60-minute pre-incubation of the GRK-2 inhibitor of Example 4 on the translocation of morphine-induced p-arrestine from the opioid receptor | i using a Transfluor® assay.

"Alkyl" refers to a saturated aliphatic hydrocarbon that includes straight chain and branched chain groups. "Alkyl" can be exemplified by groups such as methyl, ethyl, n-propyl, isopropyl and n-butyl. The alkyl groups may be substituted or unsubstituted. When substituted, the substituent group is preferably amino, cyano, halogen, alkoxy or hydroxyl. "C1-C4 alkyl" refers to alkyl groups containing one to four carbon atoms.

"Acyl" refers to the group -C (O) R in which R is C1-C4 alkyl, aryl, heteroaryl, Ci-C4-aryl alkyl or C1-C4-heteroaryl alkyl.

"Alkoxy" refers to the group -O-alkyl in which the alkyl has the definition given above.

"Carboxyl" refers to the group -C (= O) O-C1-C4 alkyl.

"Carbonylamino" refers to the group -C (O) NR'R 'in which each R' is independently hydrogen, C1-C4 alkyl, aryl, heteroaryl, C1-C4 alkyl-aryl or C1-C4 alkyl-heteroaryl .

"Aryl" refers to an aromatic carbodyl group. "Aryl" can be exemplified by phenyl. The aryl group may be substituted or unsubstituted. When substituted, the substituent group is preferably amino, cyano, halogen or hydroxyl.

"C1-C4 alkyl-aryl" refers to C1-C4 alkyl groups having an aryl substituent so that the aryl substituent is attached through an alkyl group. "C1-C4 alkyl-aryl" can be exemplified by benzyl.

"C1-C4 alkyl-heteroaryl" refers to C1-C4 alkyl groups having a heteroaryl substituent so that the heteroaryl substituent is attached through the alkyl group.

"Halogen" refers to fluorine, chlorine, bromine or iodine atoms.

"Heteroaryl" refers to a mono or disubstituted monodyl aromatic carbodyl radical having one or more heteroatoms in the carbodyl ring, in which the substituents can be halogen, cyano, nitro or C1-C4 alkyl.

"Thioalkyl" refers to the group -S-alkyl.

"Sulfonyl" refers to the group -S (O) 2R 'in which R' is hydrogen, C1-C4 alkyl, aryl, heteroaryl, C1-C4 alkyl-aryl or C1-C4 alkyl-heteroaryl.

"Sulfonylamino" refers to the group -S (O) 2NH-.

"Pharmaceutically acceptable carrier" means a carrier that is useful for the preparation of a pharmaceutical composition that is generally compatible with the other constituents of the composition, not harmful to the recipient, and neither biologically nor otherwise undesirable. "A pharmaceutically acceptable carrier" includes both one and more than one carrier. Embodiments include carriers for topical, ocular, parenteral, intravenous, intraperitoneal, intramuscular, sublingual, nasal and oral administration. "Pharmaceutically acceptable carrier" also includes agents for the preparation of aqueous dispersions and sterile powders for injection or dispersions.

"Excipient" as used herein includes physiologically compatible additives useful in the preparation of a pharmaceutical composition. Examples of pharmaceutically acceptable carriers and excipients can be found in Remington Pharmaceutical Science, 16th ed.

"Therapeutically effective amount" as used herein refers to a dosage of the compounds or compositions, effective in influencing, reducing, inhibiting or preventing desensitization of a receptor, particularly GPCR desensitization. This term as used herein may also refer to an amount effective to cause a desired in vivo effect in an animal, preferably, a human being, such as reduction in intraocular pressure.

"Administering" as used herein refers to the administration of the compounds as necessary to achieve the desired effect.

"Eye disease" as used herein includes glaucoma, allergy and dry eye.

The term "disease or condition associated with G protein receptor kinase activity" is used to mean a

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disease or condition that results, in whole or in part, from the effect on GPCR of one or more GRK.

The term "control the disease or condition" is used to mean changing the effect on GPCR of one or more GRKs to affect the disease or condition.

"Desensitization" or "GPCR desensitization" generally refers to the process by which sensitized GPCRs become desensitized GPCRs.

"Desensitized GPCR" means a GPCR that currently has no capacity to respond to an agonist and activate the conventional G-protein signaling.

"Sensitized GPCR" means a GPCR that currently has the capacity to respond to an agonist and activate conventional G-protein signaling.

"GPCR desensitization route" means any cellular component of the GPCR desensitization process, as well as any cellular structure involved in the GPCR desensitization process and subsequent processes, including arrests, GRK, GPCR, AP-2 proteina, clatrine and protema phosphatases

"GPCR signaling" means GPCR-induced activation of G proteins. This may result in, for example, cAMP production.

"G protein-coupled receptor kinase" (GRK) includes any kinase that has the ability to phosphorylate a GPCR.

"GPCR desensitization activity" of a composition (eg, compound, dissolution, etc.) means that the composition can inhibit GPCR desensitization of at least one specific GPCR.

The term "inhibit the protein kinase receptor activity G" or "inhibit the action of a GRK" means reducing or decreasing the action of the GRK.

The term "influence the GRK activity" or "influence the action of the GRK" means changing or affecting the action or activity of a GRK in one or more GPCRs.

In a preferred embodiment of formula (I), A is an unsubstituted isoquinoline radical and R1, R3 and R5 are hydrogen.

In another preferred embodiment of formula (I), R1, R3 and R5 are hydrogen and R2 'or R4' is -O-R, as defined above.

In some preferred embodiments, isoquinolines include those compounds in which R1 ’

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R3 'and R5' are

hydrogen and X ’is n. In further preferred embodiments, one of R or R is also hydrogen. In some preferred embodiments, either R2 or R4 'is hydrogen and the other group R2' or R4 'is -O-benzyl; cyano; -C (O) -NH-phenyl; hydrogen; -O-phenyl; -S-C1-C4 alkyl, preferably -S-CH3; -C1-C4 alkyl, preferably methyl; -C (O) -NH-m-pyridine; -C (O-) NH2; -SO2-NH2; -C (O) O-C1-C4 alkyl, preferably -C (O) O-CH3; -C (O) phenyl; -C (O) NH-C1-C4alkyl, preferably -C (O) NH-CH3; halogen, preferably chlorine or fluorine; -C (O) -C1-C4 alkyl, preferably -C (O) -CH3; or -O-C1-C4 alkyl, preferably O-CH3 or O-propyl, preferably isopropyl. In some preferred embodiments R2 'and R4' are hydrogen and R3 is selected from the group comprising -O-benzyl; cyano; -C (O) -NH-phenyl; hydrogen; -O-phenyl; -S-C1-C4 alkyl, preferably -S-CH3; -C1-C4 alkyl, preferably methyl, -C (O) -NH-m-pyridine, -C (O-) NH2, -SO2-NH2, -C (O) O-C1-C4 alkyl, preferably -C ( O) O-CH3; -C (O) -aryl; -C (O) NH-C1-C4 alkyl, preferably -C (O) NH-CH3; halogen; -C (O) -C1-C4 alkyl, preferably -C (O) -Ch3; or -O-C1-C4 alkyl, preferably O-CH3 or O-propyl, preferably isopropyl.

Isoquinoline compounds can be synthesized by the general schemes set out below:

Isoquinoline Synthesis

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Scheme 1

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Scheme 1: The corresponding substituted aniline was treated with a suitable protecting group such as tert-butoxycarbonyl, followed by reaction with a suitable electrophile in an aprotic solvent in the presence of an appropriate base such as NaH. Saponification of the glycine ester derivative produces the appropriate carboxylic acid, which can be coupled to 6-aminoisoquinoline using conventional amide coupling procedures to provide the substituted aminoisoquinoline. Elimination of the protective group following established protocols for such transformations provides the final isoquinoline derivatives.

Scheme 2

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Scheme 2: Alternatively, compounds of the invention can be prepared by the method described in Scheme 2. A 6-aminoisoquinoline can be acylated using the appropriate acylating agent in an aprotic solvent with an appropriate base. The use of LDA as a base and chloroacetyl chloride provides a chloroacetamide derivative. Treatment of this material with the corresponding substituted aniline in an appropriate solvent and optionally heating the reaction provide the final isoquinoline derivatives.

The group Ra generally represents the substituents set forth in either formula I or formula II for groups R1, R2, R3, R4 and R5 or R1 ', R2', R3 ', R4' and R5 '. Abbreviations used in the synthetic schemes shown

They have the following meanings: BoC2O means di-tert-butyl dicarbonate, HATU means 2- (7-aza-1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate, LDA means lithium diisopropylamide , DMF is dimethylformamide and THF is tetrahydrofuran.

The isoquinoline compounds of formula (I) or formula (II) and the compositions that include them have inhibitory activity of GPCR desensitization and can be useful in the influence or inhibition of the action of G protein receptor kinases, the influence , the prevention or inhibition of desensitization of phosphorylated receptors by G-protein receptor kinases, the influence or inhibition of other GRK-mediated events and in the treatment and / or prevention of diseases or conditions controlled by receptors affected by one or more more than the protein G receptor kinases. Isoquinolines can be used to influence or inhibit the action of GRK either in an in vitro cell or in a cell in a living body in vivo. Specifically, in one embodiment, there is provided a method of inhibiting the action of a receptor kinase coupled to G-proteins that comprises applying to a medium such as a test medium or contacting a cell or in an in vitro cell or either in a cell in a living body in vivo an effective inhibitory amount of a compound according to formula (I) or (II). In a preferred embodiment, the inhibited GRK is GRK-2, GRK-3, GRK-5 or GrK-6. In a further preferred embodiment, the inhibited GRK is GRK-2.

The present specification discloses that isoquinolines are used according to formulas I or II in methods of reducing the desensitization of GPCR in a cell comprising administering to, or contacting, the cell with a therapeutically effective amount of one or more of isoquinolines. The one or more of the isoquinolines are preferably administered in a pharmaceutically acceptable formulation, such as in or with a pharmaceutically acceptable carrier when the isoquinolines are administered to a cell or cells in a living body or organism. In another embodiment, the isoquinolines according to formulas I or II are used in methods to influence the action of a receptor kinase coupled to G-proteins in a cell comprising administering to, or contacting, the cell an effective amount of a or more isoquinolines to influence the action of GRK in the cell. The one or more of the isoquinolines are preferably administered in a pharmaceutically acceptable formulation, such as in or with a pharmaceutically acceptable carrier when the isoquinolines are administered to

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a cell or cells in a living body or organism.

Treatment or prevention of diseases or conditions for which isoquinolines may be useful include any of the diseases or conditions associated with the receptor kinase activity of G-proteins or diseases or conditions affected by GRK-mediated desensitization of GPCRs. As an example, continuous exposure to endogenous stimuli can cause regulation by reduction and loss of beneficial GPCR response in certain hereditary diseases as well as most chronic diseases. Examples of this type of pathological behavior include regulation by reduction and loss of response by both p-1 and p-2 adrenergic receptors in congestive heart failure. Desensitization is also observed by regulation by reduction of the receptors with the exogenous administration of agonists or drugs such as morphine for pain or salbutamol for asthma, for example, in which desensitization of the receptors results in an undesirable adverse effect. known as drug tolerance. Isoquinolines can be used to influence or reduce GRK controlled desensitization for states affected by the action or activity of GRKs, resulting in a therapeutic effect.

The isoquinolines in some embodiments will be administered together with the administration of a therapeutic agent that is intended to influence or control receptors coupled to specific G-proteins for the treatment or prevention of a condition or disease affected by those specific receptors. The combination of the administration of isoquinolines with a therapeutic agent directed to GPCR will provide a reduction or prevention of desensitization of the receptors to which the therapeutic agent is directed, resulting in the improvement of the ability of the therapeutic agent to have the effect. desired over a longer period of time. Additionally, administration of the therapeutic agent or receptor agonist with an isoquinoline formulation will allow lower doses of the therapeutic agent to be administered for a longer period of time.

One or more therapeutic agents can be administered with one or more isoquinoline compounds. The therapeutic agents and / or isoquinoline compounds are preferably administered in a pharmaceutically acceptable formulation with a pharmaceutically acceptable carrier when the isoquinolines are administered to a cell or cells in a living body or organism.

Compositions that include the isoquinolines of formulas I or II can be obtained in the form of various salts or solvates. As salts, physiologically acceptable salts or salts available as raw materials are used.

Pharmaceutical compositions can be formulated for use according to the present invention in a conventional manner using one or more pharmaceutically acceptable carriers or excipients. Thus, the compounds and their pharmaceutically acceptable solvates and salts can be formulated for administration by, for example, eye drops, in a formulation based on topical oil, injection, inhalation (either through the mouth or through the nose), oral, oral, parenteral or rectal administration. Techniques and formulations can generally be found in "Remington's Pharmaceutical Sciences," (Meade Publishing Co., Easton, Pa.). The therapeutic compositions should normally be sterile and stable under the conditions of manufacture and storage.

The compositions of the present invention may comprise a safe and effective amount of the subject compounds, and a pharmaceutically acceptable carrier. As used herein, "safe and effective amount" means an amount of a compound sufficient to significantly induce a positive modification in the condition to be treated, but low enough to avoid serious side effects (at a risk reason / reasonable benefit), within the scope of the founded medical criteria. A safe and effective amount of a compound will vary with the particular condition being treated, the age and physical condition of the patient being treated, the severity of the condition, the duration of the treatment, the nature of the concurrent therapy, the pharmaceutically acceptable carrier. particular used and similar factors within the knowledge and experience of the responsible physician.

The route by which the compounds of the present invention (component A) will be administered and the form of the composition will dictate the type of carrier (component B) to be used. The composition may be in a variety of forms, suitable, for example, for systemic administration (for example, oral, rectal, nasal, sublingual, buccal, implants or parenteral) or topical administration (for example, local application on the skin, eye, liposome delivery systems or iontophoresis).

The carriers for systemic administration normally comprise at least one of a) diluents, b) lubricants, c) binders, d) disintegrants, e) dyes, f) aromas, g) sweeteners, h) antioxidants, j) preservatives, k) glidants, m) solvents, n) suspending agents, or) wetting agents, p) surfactants, combinations thereof, and others. All carriers are optional in the systematic compositions.

Constituent a) is a diluent. Suitable diluents for solid dosage forms include sugars such as glucose, lactose, dextrose and sucrose; diols such as propylene glycol; calcium carbonate; sodium carbonate; sugar alcohols, such as glycerin; Mannitol and sorbitol. The amount of constituent a) in the systemic composition is usually from about 50 to about 90%.

Constituent b) is a lubricant. Suitable lubricants for solid dosage forms are exemplified

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by solid lubricants including silica, talc, stearic acid and its magnesium and calcium salts, calcium sulfate; and liquid lubricants such as polyethylene glycol and vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and theobroma oil. The amount of constituent b) in the systemic composition is usually from about 5 to about 10%.

The constituent c) is a binder. Suitable binders for solid dosage forms include polyvinylpyrrolidone; magnesium aluminum silicate; starches such as corn starch and potato starch; jelly; gum tragacanth; and cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, methyl cellulose, microcrystalline cellulose and sodium carboxymethyl cellulose. The amount of constituent c) in the systemic composition is usually from about 5 to about 50%.

The constituent d) is a disintegrant. Suitable disintegrants for solid dosage forms include agar, alchemical acid and sodium salt thereof, effervescent mixtures, croscarmellose, crospovidone, sodium carboxymethyl starch, sodium starch glycolate, clays and ion exchange resins. The amount of constituent d) in the systemic composition is usually from about 0.1 to about 10%.

The constituent e) for solid dosage forms is a dye such as an FD&C dye. The amount of constituent e) in the systemic composition is usually from about 0.005 to about 0.1%.

The constituent f) for solid dosage forms is an aroma such as menthol, peppermint and fruit aromas. The amount of constituent f) in the systemic composition is usually from about 0.1 to about 1.0%.

Constituent g) for solid dosage forms is a sweetener such as aspartame and saccharin. The amount of constituent g) in the systemic composition is usually from about 0.001 to about 1%.

The constituent h) is an antioxidant such as butylated hydroxyanisole ("BHA"), butylated hydroxytoluene ("BHT") and vitamin E. The amount of constituent h) in the systemic composition is usually from about 0.1 to about 5. %.

Constituent j) is a preservative such as benzalkonium chloride, methylparaben and sodium benzoate. The amount of constituent j) in the systemic composition is usually from about 0.01 to about 5%.

The constituent k) for solid dosage forms is a slider such as silicon dioxide. The amount of constituent k) in the systemic composition is usually from about 1 to about 5%.

The constituent m) is a solvent, such as water, isotonic saline solution, ethyl oleate, alcohols such as ethanol and phosphate buffer solutions. The amount of constituent m) in the systemic composition is usually from about 0 to about 100%.

Constituent n) is a suspending agent. Suitable suspending agents include AVICEL® RC-591 (from FMC Corporation of Philadelphia, PA) and sodium alginate. The amount of constituent n) in the systemic composition is usually from about 1 to about 8%.

Constituent o) is a surfactant such as lecithin, Polysorbate 80 and sodium lauryl sulfate, and the TWEEN® from Atlas Powder Company of Wilmington, Delaware. Suitable surfactants include those disclosed in the C.T.F.A. Cosmetic Ingredient Handbook, 1992, pags. 587-592; Remington's Pharmaceutical Sciences, 15th Ed. 1975, pgs. 335-337; and McCutcheon's Volume 1, Emulsifiers & Detergents, 1994, Edition for North America, pags. 236239. The amount of constituent o) in the systemic composition is normally from about 0.1% to about 2%.

Although the amounts of components A and B in the systemic compositions will vary depending on the type of systemic composition prepared, the specific derivative selected for component A and the constituents of component B, in general, the system compositions comprise from 0.01% to 50% of component A and 50 to 99.99% of component B.

Compositions for parenteral administration typically comprise A) from 0.01 to 10% of the compounds of the present invention and B) from 90 to about 99.9% of a carrier comprising a) a diluent and m) a solvent. In one embodiment, constituent a) comprises propylene glycol and m) comprises ethanol or ethyl oleate.

Compositions for oral administration may have various dosage forms. For example, solid forms include tablets, capsules, granules and bulk powders. These oral dosage forms comprise a safe and effective amount, usually at least about 5%, and more particularly

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from about 25% to about 50% of component A). The oral dosage compositions further comprise from about 50% to about 95% of component B), and more particularly, from about 50% to about 75%.

The tablets can be compressed, crushed into tablets, coated with enteric coating, coated with sugar, coated with film or compressed multiple times. The tablets normally comprise component A and component B, a carrier comprising constituents selected from the group consisting of a) diluents, b) lubricants, c) binders, d) disintegrants, e) dyes, f) aromas, g) sweeteners, k ) sliders and combinations thereof. Specific diluents include calcium carbonate, sodium carbonate, mannitol, lactose and cellulose. Specific binders include starch, gelatin and sucrose. Specific disintegrants include almic acid and croscarmellose. Specific lubricants include magnesium stearate, stearic acid and talc. Specific dyes are FD&C dyes, which can be added to the appearance. Chewable tablets preferably contain g) sweeteners such as aspartame and saccharin, or f) aromas such as menthol, peppermint, fruit aromas or a combination thereof.

Capsules (including programmed release and sustained release formulations) typically comprise component A, and a carrier comprising one or more a) diluents disclosed above in a capsule comprising gelatin. The granules normally comprise component A, and preferably also comprise k) sliders such as silicon dioxide to improve flow characteristics.

The selection of constituents in the carrier for oral compositions depends on secondary considerations such as taste, cost and storage stability, which are not critical for the purposes of this invention. One skilled in the art knows how to select appropriate constituents without too much experimentation.

Solid compositions can also be coated by conventional methods, usually with coatings dependent on pH or time, so that component A is released into the gastrointestinal tract in the vicinity of the desired application, or at various points and times to prolong the action desired. The coatings normally comprise one or more components selected from the group consisting of cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropylmethyl cellulose phthalate, ethyl cellulose, EUDRAGIT® coatings (available from Rohm & Haas GMBH of Darmstadt, Germany) , waxes and shellac.

Compositions for oral administration may also have liquid forms. For example, suitable liquid forms include aqueous solutions, emulsions, suspensions, reconstituted solutions from non-effervescent granules, reconstituted suspensions from non-effervescent granules, effervescent preparations reconstituted from effervescent granules, elixirs, tinctures and syrups. Liquid compositions administered orally normally comprise component A and component B, namely, a carrier comprising constituents selected from the group consisting of a) diluents, e) dyes, f) aromas, g) sweeteners, j) preservatives, m) solvents, n) suspending agents and i) surfactants. The liquid peroral compositions preferably comprise one or more constituents selected from the group consisting of e) dyes, f) aromas and g) sweeteners.

Other compositions useful for achieving systematic administration of the subject compounds include sublingual, oral and nasal dosage forms. Such compositions normally comprise one or more of soluble fillers such as a) diluents including sucrose, sorbitol and mannitol; and c) binders such as gum arabic, microcrystalline cellulose, carboxymethyl cellulose and hydroxypropyl methylcellulose. Such compositions may further comprise b) lubricants, e) colorants, f) aromas, g) sweeteners, h) antioxidants and k) glides.

In one embodiment of the invention, the compounds of the present invention are administered topically. Topical compositions that can be applied locally to the eyes may be in any form known in the art, examples of which include gellable drops, aerosols, ointments or a sustained or unsupported release unit placed at the bottom of the conjunctival sac of the eye.

Topical compositions that can be applied locally to the skin can be in any form including solutions, oils, creams, ointments, gels, lotions, shampoos, conditioners without rinse (leave-on) and with rinse (rinse-out), milks, cleansers , moisturizers, sprays and skin patches. Topical compositions comprise: component A, the compounds described above, and component B, a carrier. The carrier of the topical composition preferably aids in the penetration of the compounds in the eye. Component B may also comprise one or more optional components.

The dosage range of the compound for systemic administration is from about 0.01 to 1000 µg / kg body weight, preferably from about 0.01 to 100 µg / kg body weight, most preferably from about 1 to 50 | ig / kg body weight per day. Transdermal dosages will be designed to obtain similar serum or plasma levels, based on techniques known to those skilled in the art of pharmacokinetics and transdermal formulations. Plasma levels for transdermal administration are expected to be in the range of 0.01 to 100 nanograms / ml, more preferably

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from 0.05 to 50 ng / ml and most preferably from 0.01 to 10 ng / ml. Although these dosages are based on the daily administration rate, accumulated weekly or monthly dosages can also be used to calculate the weather requirements.

Dosages may vary based on the patient being treated, the condition being treated, the severity of the condition being treated, the route of administration, etc. to achieve the desired effect.

The compounds of the present invention are also useful in decreasing intraocular pressure. Therefore, these compounds are useful in the treatment of glaucoma. The preferred route of administration to treat glaucoma is topically.

The exact amounts of each component in the topical composition depend on several factors. The amount of component A added to the topical composition depends on the IC50 of component A, normally expressed in nanomolar units (nM). For example, if the IC50 of the drug is 1 nM, the amount of component A will be from about 0.0001 to about 0.01%. If the IC50 of the drug is 10 nM, the amount of component A) will be from about 0.001 to about 0.1%. If the IC50 of the drug is 100 nM, the amount of component A will be from about 0.01 to about 1.0%. If the IC50 of the drug is 1000 nM, the amount of component A will be 0.1 to 10%, preferably 0.5 to 5.0%. If the amount of component A is outside the ranges specified above (ie, either greater or lesser), the effectiveness of the treatment can be reduced. The IC 50 can be calculated according to the method in reference example 1, below. A person skilled in the art will know how to calculate an IC 50. The rest of the composition, up to 100%, is component B.

The amount of the carrier used together with component A is sufficient to provide a practical amount of composition for administration by unit dose of the drug. Techniques and compositions for preparing useful dosage forms in the compounds for use of this invention are described in the following references: Modern Pharmaceutics, Chapters 9 and 10, Banker & Rhodes, eds. (1979); Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1981); and Ansel, Introduction to Pharmaceutical Dosage Forms, 2nd Ed., (1976).

Component B may comprise a single constituent or a combination of two or more constituents. In topical compositions, component B comprises a topical carrier. Suitable topical carriers comprise one or more constituents selected from the group consisting of phosphate buffered saline solution, isotonic water, deionized water, monofunctional alcohols, symmetric alcohols, Aloe vera gel, alantoma, glycerin, oils with vitamin A and E, oil mineral, propylene glycol, PPG-2 myristyl propionate, dimethyl isosorbide, castor oil and combinations thereof. More particularly, carriers for cutaneous applications include propylene glycol, dimethyl isosorbide and water, and even more particularly, phosphate buffered saline, isotonic water, deionized water, monofunctional alcohols and symmetric alcohols.

The carrier of the topical composition may further comprise one or more constituents selected from the group consisting of q) emollients, r) propellants, s) solvents, t) humectants, u) thickeners, v) powders, w) fragrances, x) pigments ey) preservatives.

The constituent q) is an emollient. The amount of constituent q) in a topical skin-based composition is usually from about 5% to about 95%. Suitable emollients include steanolic alcohol, glyceryl monorricinoleate, glyceryl monostearate, propane-1,2-diol, butane-1,3-diol, mink oil, cetyl alcohol, isopropyl isostearate, stearic acid, isobutyl palmitate, stearate of isocetyl, olefinic alcohol, isopropyl laurate, hexyl laurate, decyl oleate, octadecan-2-ol, isocetyl alcohol, cetyl palmitate, di-n-butyl sebacate, isopropyl myristate, isopropyl palmitate, isopropyl stearate , butyl stearate, polyethylene glycol, triethylene glycol, lanolin, sesame oil, coconut oil, mamma oil, castor oil, acetylated lanolin alcohols, petroleum, mineral oil, butyl myristate, isostearic acid, palmic acid, isopropyl linoleate , lauryl lactate, myristyl lactate, decyl oleate, myristyl myristate and combinations thereof. Skin specific emollients include steanolic alcohol and polydimethylsiloxane.

The constituent r) is a propellant. The amount of constituent r) in the topical composition is usually from about 0% to about 95%. Suitable propellants include propane, butane, isobutane, dimethyl ether, carbon dioxide, nitrous oxide and combinations thereof.

The constituent s) is a solvent. The amount of the constituent s) in the topical composition is usually from about 0% to about 95%. Suitable solvents include water, ethyl alcohol, methylene chloride, isopropanol, castor oil, ethylene glycol monoethyl ether, diethylene glycol monobutyl ether, diethylene glycol monoethyl ether, dimethyl sulfoxide, dimethylformamide, tetrahydrofuran and combinations thereof. Specific solvents include ethyl alcohol and homotopic alcohols.

The constituent t) is a humectant. The amount of constituent t) in the topical composition is usually from 0% to 95%. Suitable humectants include glycerin, sorbitol, sodium 2-pyrrolidone-5-carboxylate, soluble collagen, dibutyl phthalate, gelatin and combinations thereof. Specific moisturizers include glycerin.

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The constituent u) is a thickener. The amount of constituent u) in the topical composition is usually from about 0% to about 95%.

The constituent v) is a powder. The amount of constituent v) in the topical composition is normally from 0% to 95%. Suitable powders include beta-cyclodextrins, hydroxypropylcyclodextrins, crete, talc, batan earth, caolm, starch, gums, colloidal silicon dioxide, sodium polyacrylate, tetraalkylammonium smectites, triaalkylarylammonium smectites, aluminum silicate, and chemically modified magnesium of organically modified montmorillonite, hydrated aluminum silicate, pyrogenic silica, carboxyvinyl polymer, sodium carboxymethylcellulose, ethylene glycol monostearate and combinations thereof. For ocular applications, specific powders include beta-cyclodextrin, hydroxypropylcyclodextrin and sodium polyacrylate. For ocular gel dosage formulations, sodium polyacrylate can be used.

The constituent w) is a fragrance. The amount of constituent w) in the topical composition is usually from about 0% to about 0.5%, particularly from about 0.001% to about 0.1%. For eye applications, a fragrance is not normally used.

The constituent x) is a pigment. Suitable pigments for cutaneous applications include inorganic pigments, organic lacquer pigments, pearly pigments and mixtures thereof. The inorganic pigments useful in this invention include those selected from the group consisting of rutile or anatase-type titanium dioxide, encoded in the Color index with reference CI 77.891; black, yellow, red and brown iron oxides, encoded with CI references 77.499, 77.492 and 77.491; manganese violet (CI 77,742); ultramarine blue (CI 77.007); chromium oxide (CI 77.288); chromium hydrate (CI 77.289); and ferric blue (Cl 77.510) and mixtures thereof.

The lacquers and organic pigments useful in this invention include those selected from the group consisting of D&C red No. 19 (CI 45,170), D&C red No. 9 (CI 15,585), D&C red No. 21 (CI 45,380) , D&C Orange No. 4 (CI 15,510), D&C Orange No. 5 (CI 45,370), D&C Red No. 27 (CI 45,410), D&C Red No. 13 (CI 15,630), D&C Red No. 7 (CI 15,850), D&C red No. 6 (CI 15,850), D&C yellow No. 5 (CI 19,140), D&C red No. 36 (CI 12,085), D&C orange No. 10 (CI 45,425), D&C yellow No. 6 (CI 15,985), D&C red No. 30 (CI 73,360), D&C red No. 3 (CI 45,430), coloring matter or lacquers based on cochineal carmm (CI 75,570) and mixtures thereof.

The pearly pigments useful in this invention include those selected from the group consisting of white pearly pigments such as mica coated with titanium oxide, bismuth oxychloride, colored pearl pigments such as titanium mica with iron oxides, titanium mica with blue ferric and chromium oxide, titanium mica with an organic pigment of the type mentioned above as well as those based on bismuth oxychloride and mixtures thereof. The amount of pigment in the topical composition is usually from about 0% to about 10%. For eye applications, a pigment is generally not used.

In a particularly preferred embodiment of the invention, topical pharmaceutical compositions for ocular administration are prepared which normally comprise component A and B (a carrier), such as purified water, and one or more constituents selected from the group consisting of y) sugars or sugar alcohols such as dextrans, particularly mannitol and dextran 70, z) cellulose or a derivative thereof, aa) a salt, bb) disodium EDTA (disodium edetate) and cc) a pH adjustment additive.

Examples of z) cellulose derivatives suitable for use in the topical pharmaceutical composition for ocular administration include sodium carboxymethyl cellulose, ethyl cellulose, methyl cellulose and hydroxypropyl methyl cellulose, particularly hydroxypropyl methyl cellulose.

Examples of aa) salts suitable for use in the topical pharmaceutical composition for ocular administration include mono, di and trisodium phosphate, sodium chloride, potassium chloride and combinations thereof.

Examples of cc) pH adjustment additives include HCl or NaOH in amounts sufficient to adjust the pH of the topical pharmaceutical composition for ocular administration to 6.8-7.5.

Component A may be included in kits comprising component A, a systematic or topical composition described above, or both; and information, instructions, or both, providing the use of the treatment kit for medical and cosmetic conditions in the birds (particularly, humans). The information and instructions may be in the form of words, pictures or both. In addition, or alternatively, the kit may comprise the medicament, a composition, or both; and information, instructions, or both, concerning the methods of application of the medicament, or of the composition, preferably with the benefit of treating or preventing medical and cosmetic conditions in mares (for example, human beings).

The invention will be further explained by the following illustrative examples.

Procedures for the preparation of isoquinolines are described in the following examples.

All temperatures are given in degrees Celsius. Reagents were purchased from commercial sources or were

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they prepared following bibliography procedures.

Unless otherwise indicated, HPLC purification was performed by redissolving the residue in a small volume of DMSO and filtering through a 0.45 micrometer syringe filter (nylon disc). The solution was then purified using a 50 mm Varian Dynamax Microsorb Guard-8 Ca column for 21.4 mm HPLC. The initial concentration of 40-80% MeOH / H2O was selected as it was appropriate for the target compound. This initial gradient was maintained for 0.5 minutes, then increased to 100% MeOH: 0% H2O over 5 minutes. 100% MeOH was maintained for another 2 minutes before rebalancing to the initial starting gradient. The total execution time was 8 minutes. The resulting fractions were analyzed, combined as appropriate and then evaporated to provide purified material.

Proton magnetic resonance spectra (1H-NMR) were recorded in any of a Varian (1H) -RMN INOVA 400 MHz spectrophotometer, Varian (1H) -RMN INOVA 500 MHz spectrophotometer, (1H) -RMN spectrophotometer Bruker, Bruker (1H) -RMN DPX 400 MHz spectrophotometer or Bruker (1H) -RMN DRX 500 MHz spectrophotometer. All spectra were determined in the solvents indicated. Although chemical shifts in ppm are reported at low field with respect to tetramethylsilane, they refer to the residual proton peak of the respective solvent peak for 1 H-NMR. Interprotonic coupling constants are reported in Hertz (Hz). Analytical HPLC was performed using a 5 micrometer Cia 125 A 50 x 4.60 mm Aqua column of Phenomenex coupled with a VWD UV detector of the 1100 series of Agilent. A neutral 0.1% (w / v) BES buffer of pH 7.1 with LiOH and 1% CH3CN in H2O is used as the aqueous phase. The initial gradient was 55% MeOH aqueous buffer that was increased to 100% MeOH over 3 minutes. 100% MeOH was maintained for 2 minutes before rebalancing to the initial starting gradient. The spectra were analyzed at 254 nm. The CL-MS spectra were obtained using an AQA MS ESI instrument from Thermofinnigan. Samples were passed through a 5 micrometer C18 125 A 50 x 4.60 mm Aqua column of Phenomenex. The initial gradient was 55% MeOH: 1% CH3CN in H2O which was increased to 100% MeOH over 3 minutes. 100% MeOH was maintained for 2 minutes before rebalancing to the initial starting gradient. The spray setting of the EM probe was at 350 µl / min with a cone voltage at 25 mV and a probe temperature at 450 ° C.

The following preparations illustrate procedures for the preparation of intermediates and methods for the preparation of isoquinolines.

General procedure for the synthesis of isoquinolines according to scheme 2

Stage 1: Synthesis of 2-chloro-N-isoquinolin-6-yl-acetamide: A 2.0 ml vial equipped with a 6-aminoisoquinoline stir bar (100 mg, 0.7 mmol) was charged in THF ( 1.0 ml) and cooled to -78 ° C. Lithium diisopropylamide (40 µL, 0.35 mmol) was added to the reaction at -78 ° C followed by the dropwise addition of chloroacetyl chloride (62 µL, 0.7 mmol). The reaction was allowed to warm to room temperature and stirred for 30 min. The reaction was concentrated in vacuo, then the solid was triturated with cold methanol. The solid was collected by filtration to provide 2-chloro-N-isoquinolin-6-yl-acetamide (58 mg, 38%). 1H-NMR (400 MHz, DMSO-d6) 8 ppm 3.14 (s, 2H) 8.01 (dd, J = 9.08, 1.66 Hz, 1H) 8.45 (d, J = 8, 98 Hz, 2H) 8.66 (d, J = 1.56 Hz, 1H) 11.46 (s, 1H), LC-MS: 221 (M + H).

Stage 2: Synthesis of isoquinolines: A 2.0 ml vial equipped with a 2-chloro-N-isoquinolin-6-yl-acetamide stir bar was loaded (See step 1, 30 mg, 0.14 mmol) , DMF (1.0 ml), potassium iodide (70 mg, 0.42 mmol) and stirred at 45 ° C for 30 min. The corresponding substituted aniline (0.42 mmol) was added and stirred for 2 h. After completion of the reaction, it was concentrated in vacuo and the residue was purified by prep HPLC. to provide the final isoquinoline.

Example 1

image12

2- (3-Benzyloxy-phenylamino) -N-isoquinolin-6-yl-acetamide: The title compound was obtained as described in step 2: 1 H-NMR (400 MHz, methanol-d4) 8 ppm 3, 93 - 3.97 (m, 2H) 4.99 - 5.02 (m, 2H) 6.27 - 6.33 (m, 2H) 6.33 - 6.39 (m, 1H) 7.04 ( t, J = 8.09Hz, 1H) 7.16-7.41 (m, 6H) 7.68-7.76 (m, 2H) 8.02 (d, J = 8.89Hz, 1H) 8, 31-8.37 (m, 2H) 9.09 (s, 1H), LC-MS: 384 (M + H).

Example 2

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image13

N-Isoquinolin-6-yl-2- (3-methoxy-phenylamino) -acetamide: The title compound was obtained as described in step 2: 1 H-NMR (400 MHz, methanol-d4) 8 ppm 3, 71 (s, 3H) 3.95 (s, 2H) 6.21 - 6.33 (m, 2H) 7.04 (t, J = 8.00 Hz, 1H) 7.66 - 7.76 (m , 2H) 8.01 (d, J = 8.98Hz, 1H) 8.29-8.37 (m, 2H) 9.08 (s, 1H), LC-MS: 308 (M + H).

Example 3

image14

2- (3-Cyano-phenylamino) -N-isoquinolin-6-yl-acetamide: The title compound was obtained as described in step 2: 1 H-NMR (400 MHz, methanol-d4) 8 ppm 4, 04 (s, 2H) 6.92 - 7.02 (m, 3H) 7.25 - 7.34 (m, 1H) 7.75 (dd, J = 8.88, 2.05Hz, 2H) 8, 05 (d, J = 8.98Hz, 1H) 8.36 (d, J = 1.95Hz, 2H) 9.10 (s, 1H), LC-MS: 303 (M + H).

Example 4

image15

3 - [(Isoquinolin-6-ylcarbamoylmethyl) -amino1-N-phenyl-benzamide:

The title compound was obtained as described in step 2: 1H-NMR (400 MHz, methanol-d4) 8 ppm 4.07 (s, 2H) 7.09-7.14 (m, 1H) 7, 22-7.25 (m, 2H) 7.27-7.36 (m, 4H) 7.61-7.66 (m, 2H) 7.71 (d, J = 5.86Hz, 1H) 7, 75 (dd, J = 8.88, 2.05Hz, 1H) 8.03 (d, J = 8.98Hz, 1H) 8.32-8.35 (m, 1H) 8.36 (d, J = 1.95Hz, 1H) 9.09 (s, 1H), LC-MS: 397 (M + H).

Example 5

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2- (3-Chloro-phenylamino) -N-isoquinolin-6-yl-acetamide: The title compound was obtained as described in step 2: 1 H-NMR (400 MHz, methanol-d4) 8 ppm 3, 99 (s, 2H) 6.56 - 6.60 (m, 1H) 6.63 - 6.69 (m, 2H) 7.09 (t, J = 8.00 Hz, 1H) 7.70-7 , 77 (m, 2H) 8.04 (d, J = 8.98Hz, 1H) 8.32-8.37 (m, 2H) 9.09 (s, 1H), LC-MS: 312 (M + H).

Example 6

image17

N-Isoquinolin-6-yl-2-phenylamino-acetamide: The title compound was obtained as described in step 2: 1 H-NMR (400 MHz, methanol-d4) 8 ppm 3.97 (s, 2H) 6.65 - 6.75 (m, 3H) 7.10 - 7.19 (m, 2H) 7.67 - 7.78 (m, 2H) 8.02 (d, J = 8.98Hz, 1H) 8.30-8.38 (m, 2H) 9.09 (s, 1H), LC-MS: 278 (M + H).

Example 7

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N-Isoquinolin-6-yl-2- (3-phenoxy-phenylamino) -acetamide: The title compound was obtained as described in step 2: 1 H-NMR (400 MHz, methanol-d4) 8 ppm 3, 93-3.95 (m, 2H) 6.28-6.33 (m, 2H) 6.42-6.48 (m, 1H) 6.91-6.96 (m, 2H) 6.99 ( t, J = 7.32Hz, 1H) 7.08-7.15 (m, 1H) 7.19-7.25 (m, 2H) 7.69-7.74 (m, 2H) 8.03 ( d, J = 8.79Hz, 1H) 8.31-8.38 (m, 2H) 9.10 (s, 1H), LC-MS: 370 (M + H).

Example 8

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N-Isoquinolin-6-yl-2- (3-methylsulfanyl-phenylamino) -acetamide:

The title compound was obtained as described in step 2: 1H-NMR (400 MHz, methanol-d4) 8 ppm 2.40 (s, 3H) 3.97 (s, 2H) 6.43-6, 48 (m, 1H) 6.58 - 6.62 (m, 2H) 7.03 - 7.10 (m, 1H) 7.68 - 7.76 (m, 2H) 8.02 (d, J = 8.98Hz, 1H) 8.31-8.36 (m, 2H) 9.08 (s, 1H), LC-MS: 324 (M + H).

Example 9

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N-Isoquinolin-6-yl-2-m-tolylamino-acetamide: The title compound was obtained as described in step 2: 1 H-NMR (400 MHz, methanol-d4) 8 ppm 2.23 (s, 3H) 3.95 (s, 2H) 6.43-6.57 (m, 3H) 7.02 (t, J = 7.71Hz, 1H) 7.67-7.77 (m, 2H) 8, 02 (d, J = 8.98Hz, 1H) 8.30-8.37 (m, 2H) 9.08 (s, 1H), LC-MS: 292 (M + H).

Example 10

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3-r (Isoquinolin-6-ylcarbamoylmethyl) -amino1-N-pyridin-3-yl-benzamide: The title compound was obtained as described in step 2: 1 H-NMR (400 MHz, methanol-d4) 8 ppm 4.08 (s, 2H) 6.89-6.94 (m, 1H) 7.24-7.33 (m, 3H) 7.42 (dd, J = 8.40, 4.88Hz, 1H ) 7.71 (d, J = 5.86Hz, 1H) 7.75 (dd, J = 8.88, 2.05Hz, 1H) 8.03 (d, J = 8.98Hz, 1H) 8.19 - 8.24 (m, 1H) 8.28 (dd, J = 4.78, 1.46Hz, 1H) 8.33 (d, J = 5.86Hz, 1H) 8.36 (d, J = 1 , 76Hz, 1H) 8.85 (d, J = 2.54Hz, 1H) 9.09 (s, 1H), LC-MS: 398 (M + H).

Example 11

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3-Isoquinolin-6-ylcarbamoylmethyl) -amino-1-benzamide: The title compound was obtained as described in step 2: 1 H-NMR (400 MHz, methanol-d4) 8 ppm 4.04 (s, 2H) 6 , 84 - 6.88 (m, 1H) 7.15 - 7.20 (m, 2H) 7.24 (t, J = 7.91 Hz, 1H) 7.71 (d, J = 5.86Hz, 1H) 7.74 (dd, J = 8.98, 2.15Hz, 1H) 8.02 (d, J = 8.98Hz, 1H) 8.36 (t, J = 2.44Hz, 2H) 9, 09 (s, 1H), LC-MS: 321 (M + H).

Example 12

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2- (3-Isopropoxy-phenylamino) -N-isoquinolin-6-yl-acetamide: The title compound was obtained as described in step 2: 1 H-NMR (400 MHz, methanol-d4) 8 ppm 1, 24 (d, J = 6.05Hz, 6H) 3.95 (s, 2H) 4.45-4.55 (m, 1H) 6.23 (t, J = 2.25Hz, 1H) 6.25 - 6.30 (m, 2H) 7.03 (t, J = 8.10Hz, 1H) 7.69-7.76 (m, 2H) 8.02 (d, J = 8.79Hz, 1H) 8, 32 - 8.36 (m,

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2H) 9.09 (s, 1H), LC-MS: 336 (M + H).

Example 13

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N-Isoquinolin-6-yl-2- (3-sulfamoyl-phenylamino) -acetamide: The title compound was obtained as described in step 2: 1 H-NMR (400 MHz, methanol-d4) 8 ppm 4, 05 (s, 2H) 6.85 - 6.89 (m, 1H) 7.18 - 7.22 (m, 2H) 7.30 (t, J = 8.20 Hz, 1H) 7.70 (d , J = 5.86Hz, 1H) 7.74 (dd, J = 8.88, 2.05Hz, 1H) 8.03 (d, J = 8.98Hz, 1H) 8.32-8.36 (m , 2H) 9.09 (s, 1H), LC-MS: 357 (M + H).

Example 14

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3 - [(Isoquinolin-6-ylcarbamoylmethyl) -amino-1-benzoic acid methyl ester: The title compound was obtained as described in step 2: 1 H-NMR (400 MHz, methanol-d4) 8 ppm 3.84 (s, 3H) 4.04 (s, 2H) 6.89-6.94 (m, 1H) 7.25 (t, J = 7.81 Hz, 1H) 7.31-7.36 (m, 2H) 7.74 - 7.79 (m, 2H) 8.06 (d, J = 8.98Hz, 1H) 8.34 (d, J = 6.05Hz, 1H) 8.38 (d, J = I. 95Hza 1H) 9.12 (s, 1H), LC-MS: 336 (M + H).

Example 15

image26

2- (3-Benzoyl-phenylamino) -N-isoquinolin-6-yl-acetamide: The title compound was obtained as described in step 2: 1 H-NMR (400 MHz, methanol-d4) 8 ppm 4, 02 (s, 2H) 6.95 - 7.00 (m, 1H) 7.02 - 7.04 (m, 1H) 7.05 - 7.09 (m, 1H) 7.30 (t, J = 7.81 Hz, 1H) 7.35 - 7.41 (m, 2H) 7.48 - 7.55 (m, 1H) 7.69 - 7.76 (m, 4H) 8.04 (d, J = 8.98Hz, 1H) 8.33-8.37 (m, 2H) 9.10 (s, 1H), LC-MS: 382 (M + H).

Example 16

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3-r (Isoquinolin-6-ylcarbamoylmethyl) -amino1-N-methyl-benzamide:

The title compound was obtained as described in step 2: 1H-NMR (400 MHz, methanol-d4) 8 ppm 2.87 (s, 3H) 4.04 (s, 2H) 6.83 (dd, J = 8.20, 2.54Hz, 1H) 7.06-7.11 (m, 1H) 7.11-7.14 (m, 1H) 7.22 (t, J = 7.81Hz, 1H) 7.70 (d, J = 5.86Hz, 1H) 7.74 (dd, J = 8.88, 2.05Hz, 1H) 8.02 (d, J = 8.98Hz, 1H) 8.30 - 8.37 (m, 2H) 9.08 (s, 1H), LC-MS: 335 (M + H).

Example 17

image28

2- (3-Fluoro-phenylamino) -N-isoquinolin-6-yl-acetamide: The title compound was obtained as described in step 2: 1 H-NMR (400 MHz, methanol-d4) 8 ppm 3, 98 (s, 2H) 6.34 - 6.41 (m, 2H) 6.45 - 6.49 (m, 1H) 7.07 - 7.14 (m, 1H) 7.71 (d, J = 5.86Hz, 1H) 7.73 (dd, J = 8.88, 2.05Hz, 1H) 8.03 (d, J = 8.79Hz, 1H) 8.31-8.38 (m, 2H) 9.09 (s, 1 H), LC-MS: 296 (M + H).

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Example 18

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2- (3-Acetyl-phenylamino) -N-isoquinolin-6-yl-acetamide: The title compound was obtained as described in step 2: 1 H-NMR (400 MHz, methanol-d4) 8 ppm 2, 54 (s, 3H) 4.05 (s, 2H) 6.91 - 6.95 (m, 1H) 7.25 - 7.30 (m, 2H) 7.31 - 7.35 (m, 1H) 7.71 (d, J = 5.86Hz, 1H) 7.74 (dd, J = 8.88, 2.05Hz, 1H) 8.03 (d, J = 8.79Hz, 1H) 8.31 - 8.37 (m, 2H) 9.09 (s, 1H), LC-MS: 320 (M + H).

Reference Example 1:

The inhibition of receptor kinases coupled to G-proteins including hGRK-2 for isoquinoline compounds disclosed herein was determined using a biochemical assay. The inhibition of GRK-3, GRK-5 and GRK-6 was also determined using the same assay.

Protein kinase inhibition was determined using a biochemical assay that uses the emission of light from a reaction with luciferase. The luciferase-based assay works according to the following reaction principles:

GRK-2

Substrate-OH --------- ■ ----- ► Substrate-OP

ATP ^ ADP •

D-Luciferin + H202 + 02

A GRK-2 inhibitor will increase the amount of ATP in solution as shown. Therefore, a GRK-2 inhibitor will direct the reaction with luciferase to the right, resulting in more emitted light. The amount of light emitted is proportional to the inhibition that results from the GRK-2 inhibitor. The luciferase assay was also used to test the inhibition properties of other kinases. Table 1 below shows the test results for GRK-2, GRK-3, GRK-5 and GRK-6.

The test procedure was as follows:

Assay Buffer:

50 mM HEPES, pH 7.5 10 mM MgCl2

Activated sodium ortovanadate 100 pM CHAPS 0.01%

0.1% BSA

1 mM DTT (added new every day)

Stock solution of 10X assay buffer:

500 mM HEPES, pH 7.5 100 mM MgCl2

1 mM activated sodium orthovanadate 0.1% CHAPS

1% BSA (omitted for 10X compound dilution buffer)

Final test conditions:

50 pM test compound

Casema 20 pM

10 pM ATP

50 nM hGRK2

4.5% DMSO

Incubation for 90-120 minutes

It is stopped by adding 30 pl of Kinase-Glo diluted 3X reagent containing 0.01% tnpane blue. Be

image30

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30

35

40

It counts on a FUSION plate reader. PROTOCOL

Dilution of compounds and transfer

Sufficient buffer containing 40% DMSO is prepared to add 20 jLil / well to the number of plates being tested. This buffer should not contain BSA because it will precipitate with the addition of 40% DMSO. (Example: to prepare 3000 ml of compound dilution buffer: 1200 ml of DMSO, 180 ml of 10X compound dilution buffer, 1615 ml of ultrapure water, 3 ml of 1M dTt).

Using the Multidrop dispenser, 20 L of compound dilution buffer is added to all wells of the 384 well daughter plate (1 L of DMSO is added to control wells). This will result in a daughter plate containing 21 L of test compound ~ 500 | iM.

Using the PlateTrak system, 5 L of the test compounds are transferred to a white, non-union, 384-well microtiter plate (Costar XXXX).

Adding GRK2 / ATP

A sufficient volume of buffer (with 1mM BSA and DTT) containing 125 nM GRK2 and 25 µM ATP is prepared. (Example: to 219 ml of buffer, 550 L of 10 mM ATP and 350 L of GRK2 79 | iM are added).

Using the Multidrop dispenser, 20 L of GRK2 / ATP mixture is added to all wells of the microtiter plate.

Adding Casein

A sufficient volume of buffer (with 1 mM BSA and DTT) containing 40 µM casein is prepared. (Example: to 211 ml of buffer, 8.8 ml of 1 mM casein are added)

Using the Multidrop dispenser, 25 L are added to columns 1 to 23 of the microtiter plate. 25 µl of complete buffer is added to column 24 (white).

Incubation

The reaction is mixed gently by tapping (the addition with the Multidrop casein dispenser makes acceptable mixing), the plates are stacked and incubated at room temperature for 90 to 120 minutes. The track can be traced to the progress of the test on a separate plate if desired. ATp consumption of 20-30% is selected as the objective. An attempt should be made to avoid exceeding a 40% consumption since the kinematics could become non-linear due to the depletion of the substrate (ATP).

Addition of Kinase-Glo reagents

The Kinase-Glo reagent can be diluted 3 times without loss of data quality in this assay. Additionally, since the library contains many colored compounds that will extinguish the light emitted from the well resulting in a false negative (kinase inhibitors result in less ATP consumption, hence more emitted light), the entire reaction is subjected to deliberate extinction to cancel this effect. This deliberate extinction is achieved by the addition of 0.01% tnpane blue to Kinase-Glo reagents. (Example: 100 ml of Kinase-Glo 1X reagents (prepared according to the leaflet), 200 ml of assay buffer, 7.5 ml of 0.4% tnpane blue.

Using the Multidrop dispenser, 30 L are added to all wells of the test plate.

The FUSION plate reader is counted in luminescence mode.

The inhibition results for examples 1-18 above are for isoquinolines having the following general isoquinoline structure:

image31

in which Rb is given separately for each example below in Table 1. The results are provided for Ki (nM): Ki of 10-100 nM - ++++

K

K

K

100-1000 nM - 1000-10,000 nM -> 10,000 nM -

+++

++

+.

TABLE 1

 EJ

 Rb GRK-2 Inhib. max. (%) (GRK2) GRK-3 GRK-5 GRK-6

 one

 OBn + + + 112 + + + + + + +

 2

 OMe + + + + 103 + + + + + + + + + +

 3

 CN + + 91 + + - +

 4

 CONHPh + + + + 99 + + + + + + + + +

 5

 Cl + + + 102 + + + + + + +

 6

 H + + + 112 + + + + + + +

 7

 OPh + + + 104 + + + + + + +

 8

 SMe + + + + 108 + + + + + + + +

 9

 Me + + + 100 + + + + + + +

 10

 CONH-m- pyridine + + + + 106 + + + + + + + + + +

 eleven

 CONH2 + + + + 102 + + + + + + + +

 12

 O-iPr + + + + 118 + + + + + + +

 13

 SO2NH2 + + 115 + + + +

 14

 COOMe + + + 104 + + + + +

 fifteen

 COPh + + 119 + + + +

 16

 CONHMe + + + + 113 + + + + + + + +

 17

 F + + + 120 + + + + + + + +

 18

 EAT + + + 116 + + + + +

5 Example 19

An isoquinoline having the following structure is prepared as described above according to scheme 2.

image32

N-Isoquinolin-6-yl-2- (2-methoxy-phenylamino) -acetamide: The title compound such as step 2: 1 H-NMR (400 MHz, methanol-d4) 8 ppm 3.89 (s, 3H) 4.00 (s, 2H) 6.54 (dd, J = 7.81,

(m, J = 7.61, 1.56Hz, 1H) 6.78-6.83 (m, J = 7.71, 1.46Hz, 1H) 6.87 (dd, J = 7.91, 1 , 27Hz, 1H)

(d, J = 8.98Hz, 1H) 8.32-8.38 (m, 2H) 9.09 (s, 1H), LC-MS: 308 (M + H).

This isoquinoline was also tested using the biochemical test with luciferase. The maximum inhibition was 109% for GRK-2. The results in Ki (nM) using the above scale were as follows:

15 GRK-2 - ++

GRK-3 - ++

GRK-5 - +

GRK-6 - ++

Example 20

An isoquinoline having the following structure is prepared as described above according to scheme 2.

as described in 1.56Hz, 1H) 6.67-6.72 7.68-7.77 (m, 2H) 8.03

image33

N-Isoquinolin-6-yl-2- (4-methoxy-phenylamino) -acetamide: The title compound was obtained as described in step 2: 1 H-NMR (400 MHz, methanol-d4) 8 ppm 3, 69 (s, 3H) 3.91 (s, 2H) 6.61 - 6.69 (m, 2H) 6.74 - 6.82 (m, 2H) 7.67 - 7.79 (m, 2H) 8.03 (d, J = 8.79Hz, 1H) 8.30-8.38 (m, 2H) 9.09 (s, 1H), LC-MS: 308 (M + H).

5 This isoquinoline was also tested using the biochemical test with luciferase. The maximum inhibition was 116% for GRK-2. The results in Ki (nM) using the above scale were as follows:

GRK-2 - ++

GRK-3 - ++

GRK-5 - +

10 GRK-6 - ++.

Example 21

An isoquinoline having the following structure was prepared as described above according to scheme 2.

image34

15 N-Isoquinolin-6-yl-2-p-tolylamino-acetamide: The title compound was obtained as described in step 2: 1 H-NMR (400 MHz, methanol-d4) 8 ppm 2.19 (s , 3H) 3.93 (s, 2H) 6.56 - 6.63 (m, 2H) 6.97 (d, J = 8.00 Hz, 2H) 7.66 - 7.76 (m, 2H) 8.01 (d, J = 8.79Hz, 1H) 8.28-8.37 (m, 2H) 9.08 (s, 1H), LC-MS: 292 (M + H).

This isoquinoline was also tested using the biochemical test with luciferase. The maximum inhibition was 116% for GRK-2. The results in Ki (nM) using the above scale were as follows:

twenty

 GRK-2 -

 ++

 GRK-3 -

 ++

 GRK-5 -

 +

 GRK-6 -

 ++

 Example 22

An isoquinoline having the following structure was prepared as described above according to scheme 2.

image35

2- (3,5-Dimethoxy-phenylamino) -N-isoquinolin-6-yl-acetamide: The title compound was obtained as described in step 2: H-NMR (400 MHz, methanol-d4) 8 ppm 3.70 (s, 6H) 3.94 (s, 2H) 5.82-5.94 (m, 3H) 7.67-7.79 (m, 2H) 8.03 (d, J = 8, 79Hz, 1H) 8.28-8.40 (m, 2H) 9.09 (s, 1H), LC-MS: 338 (M + H).

This isoquinoline was also tested using the biochemical test with luciferase. Maximum inhibition 5 was 124% for GRK-2. The results in Ki (nM) using the above scale were as follows:

GRK-2 - ++

GRK-3 - ++

GRK-5 - ++

GRK-6 - ++.

10 Example 23 (reference example)

An isoquinoline having the following structure was prepared as described above according to scheme 2.

image36

N-Isoquinolin-6-yl-2- (3-methoxy-benzylamino) -acetamide: The title compound was obtained as described in step 2: 1 H-NMR (400 MHz, methanol-d4) 8 ppm 3 , 45 (s, 2H) 3.77 (s, 3H) 3.81 (s, 2H) 6.76-6.83 (m, 1H) 6.91-7.00 (m, 2H) 7.19 - 7.27 (m, 1H) 7.68 - 7.76 (m, 2H) 8.04 (d, J = 8.79Hz, 1H) 8.32 - 8.37 (m, 2H) 9.10 (s, 1H), LC-MS: 322 (M + H).

This isoquinoline was also tested using the biochemical test with luciferase. The maximum inhibition was 111% for GRK-2. The results in Ki (nM) using the above scale were as follows:

20 GRK-2 - ++

GRK-3 - ++

GRK-5 - +

GRK-6 - +.

Example 24 (reference example)

An isoquinoline having the following structure was prepared as described above according to scheme 2.

image37

Isoquinolin-6-yl-amide of 2,3-dihydro-1H-indole-2-carboxylic acid: A solution of 6-aminoisoquinoline (25 mg, 0.17 mmol) in DMF (1.0 ml) was added Indoline-2-carboxylic acid (30 mg, 0.17 mmol), HATU (70 mg, 30 0.19 mmol), diisopropylethylamine (35 µl, 0.19 mmol) and stirred overnight at 40 ° C. The mixture was concentrated

reaction and purified by prep HPLC. to provide the title compound (3.0 mg): 1 H-NMR (400 MHz, methanol-d4) 8 ppm 3.18 (dd, J = 16.11, 8.30 Hz, 1H) 3.56 (dd , J = 16.20, 10.54 Hz, 1H) 4.52 (dd, J = 10.54, 8.40 Hz, 1H) 6.70 - 6.81 (m, 2H) 6.99 - 7 , 10 (m, 3H) 7.73 (d, J = 6.05 Hz, 1H) 7.81 (dd, J = 8.88, 2.05 Hz, 1H) 8.05 (d, J = 8 , 98 Hz, 1H) 8.35 (d, J = 5.86 Hz, 1H) 8.40 (d, J = 1.95 Hz, 1H) 9.11 (s, 1H), LC-MS: 290 (M + H).

35 This isoquinoline was also tested using the biochemical test with luciferase. The maximum inhibition was 110% for GRK-2. The results in Ki (nM) using the above scale were as follows:

GRK-2 - ++

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40

Four. Five

GRK-3 - ++

GRK-5 - +

GRK-6 - +.

Example 25

The isoquinolines of Examples 2-4 were also examined in cell assays to determine the inhibitory effect of GRK-2. The results are shown in Figures 1-9. The figures show the effect of the three different isoquinolines on the translocation of several receptors. The Transfluor test (Assay and Drug Development Technologies, volume 1, number 1-1, pages 21-30, (2002), U.S. Patent No. 5,891,646, and U.S. Patent No. 6,110,693) was used to measure the degree of translocation in U2OS cells that overexpress the receptor and arrestine.

Figures 1-3 show the effect of the isoquinoline described in example 2 (GRK-2, Ki = 0.022 micromolar), in the translocation of isoproterenol-induced arrestine-GFP to the beta 2 adrenergic receptor (B2wt). Figure 1 shows dose-response curves for the effect of isoproterenol (a beta 2 adrenergic receptor agonist) against F grains (a measure of the degree of translocation of arrestine-GFP to the receptor) for increasing concentrations of isoquinoline (see curves) For any given concentration of isoproterenol, as the concentration of isoquinoline increases, there is a gradual reduction in translocation (decrease in F grains). By preventing GRK-2 mediated phosphorylation of the receptor, isoquinoline prevents the arrestine-GFP binding to the receptor. Figure 2 shows the same effect on the beta 1 adrenergic receptor (B1wt), and Figure 2 is the effect on the opioid receptor mu. Figures 4-6 and Figures 7-9 show the effect of two other isoquinolines, the isoquinoline of example 3 (GRK-2, Ki = 1.8 micromolar) and the isoquinoline of example 4, (GRK-2, Ki = 0.037 micromolar), respectively, in the same three receptors tested for the isoquinoline of Example 2.

The isoquinoline of example 2 and the isoquinoline of example 4, which are very good GRK-2 inhibitors show a moderate inhibition of the translocation of arrestine-GFP to B2WT, B1WT, but a strong inhibition for the opioid receptor mu. However, the isoquinoline of example 3, which is a weaker inhibitor of GRK-2, shows much less inhibition of translocation in the three receptors. Finally, increased concentrations of isoquinoline either from example 2 or from example 4 showed an increased accumulation of cAMP in HEK-293 cells (overexpressed beta 2 adrenergic receptor) in the presence of a fixed concentration of isoproterenol. This is consistent with the inhibition of GRK-2 which results in less translocation of arrestine-GFP, less desensitization, and therefore, more signaling by B2AR. With more receptors available now on the cell surface, more cAMP is being generated in the presence of Isoproterenol. The isoquinoline of example 3, which is a weak inhibitor of GRK-2 and shows much less inhibition of translocation, also has less effect on the amount of cAMP accumulated in the cell.

In Figure 1, the IC50 of p2-arrestine was 8 | iM. In Figure 3, the IC50 for the opioid receptor mu was less than 1 | iM. In Figure 4, the IC50 of p2-arrestin was greater than 100 | iM. In Figure 5, the IC50 for p1-arrestin was greater than 100 | iM. In Figure 6, the IC50 for the opioid receptor mu was greater than 100 µM. In Figure 7, the IC50 of p2-arrestine was 4 | iM. In Figure 8, the IC50 for p1-arrestin was greater than 100 | iM. In Figure 9, the IC50 for the opioid receptor mu was less than 1 | iM.

Isoquinolines can be further examined to determine the effect on desensitization of GPCR by using the methods described in U.S. Patent Application No. 2004/0091946, published May 13, 2004, U.S. Patent Application No. 2005/0032125, published on February 10, 2005.

Example 26

image38

2- (4-Benzoyl-phenylamino) -N-isoquinolin-6-yl-acetamide: The title compound could be obtained as described in step 2 above.

Reference Example 2

Swine trabecular meshwork (MTP) assay based on cells.

The previous section of pig eyes was collected within 4 hours after death. The iris and ciliary body were removed and cells from the trabecular mesh were collected by blunt dissection. Trabecular mesh fabric was seeded

finely ground in 6-well plates coated with collagen in 199 medium containing 20% fetal bovine serum (FBS). After two passes at confluence, the cells were transferred to low glucose DMEM containing 10% FBS. Cells were used between pass 3 and pass 8.

The cells were seeded in multiple glass well plates, coated with fibronectin, the day before the 5 tests with compounds under conventional culture conditions. The compounds were added to the cells in the presence of DMEm containing 1% FBS and 1% DMSO. When the compounds were incubated with the cells in the determined duration that was optimal, the media and compounds were removed and the cells were fixed for 20 minutes in 3% methanol-free paraformaldetndo. The cells were rinsed twice with phosphate buffered saline (PBS) and the cells were permeabilized with 0.5% Triton X-100 for two minutes. After 10 additional washings with pBs, F-actin was stained with phalloidin labeled with Alexa-fluor 488 and the nuclei were stained with DAPI.

The data were reduced to the mean rectilmea actin fiber length and normalized with respect to control cells treated with DMSO (100%) and Y-27632 50 | iM (0%). Y-27632 is a rho kinase inhibitor that is known to result in the depolymerization of F-actin in these cells.

15 Example 27

The cell assay described in reference example 2 was used to test examples 1-18 above, the results of which are presented below. For reference, these are isoquinolines that have the following general isoquinoline structure:

image39

20 in which Rb is provided separately for each example below in Table 2. The results regarding the activity are provided at 50 | iM compared to the control

More active than control- +++

As active as the control ++

Less active than control +

25 Inactive -

TABLE 2

 Example

 R MTP cell assay

 one

 OCH2Ph +

 2

 OMe + + +

 3

 CN + + +

 4

 CONHPh + + +

 5

 Cl + +

 6

 H + +

 7

 OPh + + +

 8

 SMe + + +

 10

 CONH-m-pyridine + + +

 14

 COOMe + + +

 fifteen

 COPh + +

Example 28

An isoquinoline having the following structure was prepared as described above according to scheme 2 set forth in example 20.

image40

This isoqumohna was also tested using the MTP cell assay as described in reference example 2 as a reference. The results using the above scale were as follows:

MTP + cell assay

5 Example 29

An isoquinoline having the following structure was prepared as described above according to scheme 2 set forth in example 21.

image41

This isoquinoline was also tested using the MTP cell assay as described in the example of reference 2. The results using the above scale were as follows:

MTP ++ cell assay

Example 30

An isoquinoline having the following structure was prepared as described above according to scheme 2 set forth in example 22.

fifteen

image42

This isoquinoline was also tested using the MTP cell assay as described in reference example 2. The results using the above scale were as follows:

MTP cell assay -

Example 31 (reference example)

An isoquinoline having the following structure was prepared as described above according to scheme 2 set forth in example 23.

image43

This isoquinoline was also tested using the MTP cell assay as described in reference example 2. The results using the above scale were as follows:

25 MTP ++ cell assay

5

10

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twenty

25

30

Reference Example 3:

Pharmacological activity for the glaucoma test.

The pharmacological activity for the glaucoma assay can be demonstrated using assays designed to test the ability of the subject compounds to decrease intraocular pressure. Examples of such tests are described in the following reference: C. Liljebris, G. Selen, B. Resul, J. Sternschantz and U. Hacksell, “Derivatives of 17-phenyl-18,19,20-trinorprostaglandin F2a Isopropyl Ester: Potential Anti-glaucoma Agents ”, Journal of Medicinal Chemistry, Vol. 38 (2) 1995, pags. 289-304.

Example 32

Topical pharmaceutical compositions are prepared to reduce intraocular pressure by conventional methods and are formulated as follows:

 Constituent

 Amount (% by weight)

 Isoquinoline derivative

 0.50

 Dextran 70

 0.1

 Hydroxypropyl methylcellulose

 0.3

 Sodium chloride

 0.77

 Potassium chloride

 0.12

 Disodium EDTA

 0.05

 Benzalkonium chloride

 0.01

 HCl and / or NaOH

 pH 7.0-7.2

 Purified water

 c.s. up to 100%

A compound according to this invention is used as the isoquinoline derivative. When the composition is topically administered to the eyes one or more times a day as drops of 40 microliters, the above composition lowers the intraocular pressure in a patient presenting glaucoma.

Example 33

Example 32 is repeated using N-isoquinolin-6-yl-2-p-tolylamino-acetamide according to this invention. When administered as a drop 4 times a day, the above composition substantially decreases intraocular pressure and serves as a neuroprotective agent.

Example 34

Example 32 is repeated using 2- (3-benzoyl-phenylamino) -N-isoquinolin-6-yl-acetamide according to this invention. When administered as a drop twice a day, the above composition substantially decreases intraocular pressure.

Example 35

Example 32 is repeated using 3 - [(isoquinolin-6-ylcarbamoylmethyl) -amino] -N-pyridin-3-yl-benzamide according to this invention. When administered as a drop twice a day, the above composition substantially decreases allergic symptoms and relieves the dry eye syndrome.

Example 36

Example 32 is repeated using a 4 - [(isoquinolin-6-ylcarbamoylmethyl) -amino] -N-pyridin-4-yl-benzamide according to this invention. When administered as a drop as necessary, the above composition substantially decreases hyperemia, redness and eye irritation.

Example 37

Example 32 is repeated using N- (isoquinolin-6-yl) -2- (4-methoxyphenylamino) acetamide according to this invention. When administered as a drop 4 times a day, the above composition substantially decreases intraocular pressure and serves as a neuroprotective agent.

Example 38

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35

Example 32 is repeated using N- (isoquinolin-6-yl) -2- (4- (methylthio) phenylamino) acetamide according to this invention. When administered as a drop twice a day, the above composition substantially decreases intraocular pressure.

Example 39

Example 32 is repeated using phenyl 3- (2- (isoquinolin-6-ylamino) -2-oxoethylamino) benzoate according to this invention. When administered as a drop twice a day, the above composition substantially decreases allergic symptoms and relieves the dry eye syndrome.

Example 40

Example 32 is repeated using phenyl 4- (2- (isoquinolin-6-ylamino) -2-oxoethylamino) benzoate according to this invention. When administered as a drop as necessary, the above composition substantially decreases allergic symptoms

Example 41

Example 32 is repeated using N- (isoquinolin-6-yl) -2- (3- (methylthio) phenylamino) acetamide according to this invention. When administered as a drop as necessary, the above composition substantially decreases hyperemia, redness and eye irritation.

Example 42

Example 32 is repeated using N- (2-fluorophenyl) -4- (2- (isoquinolin-6-ylamino) -2-oxoethylamino) benzamide according to this invention. When administered as a drop twice a day or as necessary, the above composition substantially lowers the intraocular pressure.

Example 43

Example 32 is repeated using N- (2-fluorophenyl) -2- (2- (isoquinolin-6-ylamino) -2-oxoethylamino) benzamide according to this invention. When administered as a drop twice a day or as necessary, the above composition substantially lowers the intraocular pressure.

Example 44

Example 32 is repeated using N- (isoquinolin-6-yl) -2- (3-sulfamoylphenylamino) acetamide according to this invention. When administered as a drop twice a day or as necessary, the above composition substantially lowers the intraocular pressure.

Example 45

Example 32 is repeated using N- (isoquinolin-6-yl) -2- (4-sulfamoylphenylamino) acetamide according to this invention. When administered as a drop twice a day or as necessary, the above composition substantially lowers the intraocular pressure.

Example 46

Example 32 is repeated using N- (isoquinolin-6-yl) -2- (4- (N-methylsulfamoyl) phenylamino) acetamide according to this invention. When administered as a drop twice a day or as necessary, the above composition substantially lowers the intraocular pressure.

Claims (27)

one. 5 10 2. 15 3. Four. 5. 6. twenty 7. Compound according to formula (I): image 1 wherein A is a substituted or unsubstituted isoquinolin-6-yl, in which the isoquinoline radical may be mono or disubstituted with halogen, cyano, nitro or C1-C4 alkyl; R1, R2, R3, R4 and R5 are independently hydrogen, halogen, C1-C8 alkyl, alkoxy, phenoxy, -OR7, amino, nitro, cyano, aryl, C1-C4 alkylaryl, heteroaryl, C1-C4 alkyl-heteroaryl , carbonylamino, thioalkyl, sulfonyl, sulfonylamino, acyl or carboxyl; R7 is C1-C4 alkyl, aryl, heteroaryl, C1-C4 alkyl-aryl or C1-C4 alkyl-heteroaryl; N. X is; Y R6 is CH2 or CH (C1-C4 alkyl). Compound according to claim 1, wherein R1, R2, R3, R4 and R5 are independently hydrogen, halogen, C1-C8 alkyl, alkoxy, -OR7, amino, nitro, cyano, aryl, C1-C4 alkylaryl, heteroaryl , C1-C4-heteroaryl, carbonylamino, thioalkyl, sulfonyl, sulfonylamino, acyl or carboxyl; Compound according to claim 1, wherein A is an unsubstituted isoquinoline radical and R1, R3 and R5 are hydrogen. Compound according to claim 1, wherein R1, R2, R3 and R5 are hydrogen and R4 is -O-R7 Compound according to claim 1, wherein R4 is carbonylamino, sulfylamino, acyl or carboxyl. Compound according to claim 1, wherein R1, R2 and R5 are H and one of R3 and R4 is carbonylamino, sulfylamino, acyl or carboxyl. Compound according to formula (II): image2 wherein R1 ', R2', R3 ', R4' and R5 'are, independently, hydrogen, halogen, unsubstituted C1-C4 alkyl, amino, nitro, cyano, carbonylamino, alkoxy, -O-R7, sulfonylamino, carboxyl , acyl or thioalkyl; R 7 'is C 1 -C 4 alkyl, aryl, heteroaryl, C 1 -C 4 alkyl-aryl or C 1 -C 4 alkyl-heteroaryl; X 'is image3 Y R6 is CH2 or CH (C1-C4 alkyl). Compound according to claim 7, wherein R1, R2 ', R3' and R5 'are hydrogen and R4' is -O-R7. 10 fifteen twenty 25 30 35 9. Compound according to claim 7, wherein R4 'is carbonylamino, sulfylamino, acyl or carboxyl. 10. Compound according to claim 9, wherein R4 'is carbonylamino and the carbonylamino is C (O) NH-phenyl, C (O) NH-m-pyridyl, C (O) NH-o-pyridyl, C (O ) NH-p-pyridyl, C (O) NH2 or C (O) NHCHa. 11. Compound according to claim 7, wherein R1 ', R2' and R5 'are H and one of R3' and R4 'is carbonylamino, sulfylamino, acyl or carboxyl. 12. Compound according to claim 7, wherein R2 'or R4' is -O-R7. 13. Compound according to claim 7, wherein R7 'is methyl, ethyl, phenyl, benzyl, propyl or isopropyl. 14. Compound according to formula (III): image4 wherein R8 and R9 are independently hydrogen, halogen, unsubstituted C1-C4 alkyl, substituted C1-C4 alkyl, amino, nitro, cyano, carbonylamino, alkoxy, phenoxy, benzyloxy, -O-R10, sulfonylamino, carboxyl, acyl or thioalkyl; Y R10 is unsubstituted C1-C4 alkyl, substituted C1-C4 alkyl, substituted aryl, heteroaryl, substituted heteroaryl, C1-C4 alkaryl or C1-C4-alkaryl heteroaryl, wherein when R8, R9 or R10 is substituted, the substituent is selected from amino, halogen, cyano, alkoxy or hydroxyl. 15. Compound according to claim 14, wherein R8 is carbonylamino, a carboxyl, a sulfonylamino, a cyano or an acyl moiety, and R9 is selected from H, methyl, cyano or halogen. 16. Compound according to claim 15, wherein R8 is carbonylamino, and the carbonylamino is C (O) NH-phenyl, C (O) NH-m-pyridyl, C (O) NH-o-pyridyl, C (O ) NH-p-pyridyl, C (O) NH2 and C (O) NHCH3. 17. Compound according to claim 15, wherein R8 is carboxyl and the carboxyl is C (O) O-phenyl, C (O) Om-pyridyl, C (O) Oo-pyridyl, C (O) Op-pyridyl, C (O) NH2 or C (O) OCH3. 18. Compound according to claim 15, wherein R8 is sulfonylamino, and the sulfonylamino is S (O) 2-NH-phenyl, S (O) 2-NH-m-pyridyl, S (O) 2-NH-o -pyridyl, S (O) 2-NH-p-pyridyl, S (O) 2-NH2 or S (O) 2-NHCH3. 19. Compound according to claim 14, wherein R9 is a carbonylamino, a carboxyl, a sulfonylamino, a cyano or an acyl moiety, and R8 is H, methyl, cyano or halogen. 20. Compound according to claim 14, wherein R8 is alkoxy, phenoxy, benzyloxy or -O-R10 and R9 is H, methyl, cyano or halogen. 21. Compound according to claim 14, wherein R8 and R9 are independently hydrogen, halogen, unsubstituted C1-C4 alkyl, substituted C1-C4 alkyl, amino, nitro, cyano, carbonylamino, alkoxy, benzyloxy, -O- R10, sulfonylamino, carboxyl, acyl or thioalkyl. 22. Pharmaceutical composition comprising: a) an isoquinoline derivative according to claims 1, 7 or 14; Y b) a carrier. 23. Composition according to claim 22, wherein the carrier is selected from the group consisting of systemic and topical carriers. 24. Composition according to claim 22, wherein the composition comprises approximately 0.01% to 10% of the isoquinoline derivative and 90 to 99.99% of the systemic carrier. 25. Isoquinoline derivative for use in the treatment of a state, in which the state is selected from the group consisting of eye disease, bone disorder, heart disease, liver disease, kidney disease, pancreatitis, cancer, myocardial infarction, gastric discomfort, hypertension, fertility control, nasal congestion, neurogenic bladder disorder, gastrointestinal disorder and dermatological disorder, wherein the isoquinoline derivative is of formula (I) according to claim 1. 5 26. Isoquinoline derivative for use in the treatment of a state, in which the state is selected from the group consisting of eye disease, bone disorder, heart disease, liver disease, kidney disease, pancreatitis, cancer, myocardial infarction, gastric discomfort, hypertension, fertility control, nasal congestion, bladder neurogenic disorder, gastrointestinal disorder and disorder dermatological, 10 wherein the isoquinoline derivative is of formula (II) according to claim 7. 27. Compound according to claim 25 or 26, wherein the state comprises eye disease. 28. Compound according to claim 27, wherein the state comprises glaucoma. 13 5 29. Compound according to claim 25 or 26, wherein R, R and R are independently hydrogen, halogen, Ci-Cs alkyl, alkoxy, -O-R7, amino, nitro, cyano, aryl, C1-C4 alkylaryl , heteroaryl, C1-C4 alkyl- Heteroaryl, carbonylamino, thioalkyl, sulfonyl, sulfonylamino, acyl or carboxyl; R2 and R4 are independently hydrogen, halogen, C1-C8 alkyl, alkoxy, -O-R7, amino, nitro, cyano, aryl, C1-C4 alkylaryl, C1-C4 alkyl-heteroaryl, carbonylamino, thioalkyl, sulfonyl, sulfonylamino , acyl or carboxyl. 30 twenty Compound according to claim 1.7 or 14, selected from the following: image5

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or image6 2- (3-benzyloxy-phemlamino) -N-isoquinolin-6-yl- acetamide image7 2- (3-chloro-femlammo) -N-isoquinolin-6-yl- acetamide image8 3 - [(isoqumolin-6-ylcarbamoylmethyl) -ammo] -N- phenyl benzamide J-NXjO kj H N-isoquinolin-6-yl-2- (3-methylsulfanyl- phenylamino) -acetamide image9 N-isoquinolin-6-yl-2-m-tolylamino-acetamide image10 2- (3-Isopropoxy-phenylamino) -N-isoquinolin-6- il-acetamide image11 N-isoquinolin-6-yl-2- (3-sulfamoyl-phenylamino) - acetamide image12 3 - [(isoquinolin-6-ylcarbamoylmethyl) -amino] -benzoic acid methyl ester image13 image14 2- (3-acetyl-phenylamino) -N-isoquinolin-6-yl- 2- (3-fluoro-phenylamino) -N-isoquinolin-6-yl- acetamide acetamide image15 N-isoquinolin-6-yl-2- (2-methoxy-phenylamino) - acetamide image16 N-isoquinolin-6-yl-2- (4- (methylthio) phenylamino) - acetamide image17 N- (2-fluorophenyl) -4- (2-isoquinolin-6-ylamino) - 2-oxoethylamino) benzamide image18 N- (isoquinolin-6-yl) -2- (4- sulfamoylphenylamino) acetamide image19 2- (3,5-dimethoxy-phenylamino) -N-isoquinolin-6- il-acetamide image20 4- (2- (isoquinolin-6-yl-amino) -2-oxoethylamino) phenyl benzoate image21 N- (2-fluorophenyl) -2- (2-isoquinolin-6-ylamino) - 2-oxoethylamino) benzamide image22 N- (isoquinolin-6-yl) -2- (4- (N- methylsulfamoyl) phenylamino) acetamide